1. Field of the Invention
The present invention relates to a method for preparing soluble multimeric proteins consisting of more than three iterations of the same bioactive molecule using recombinant DNA technology.
The present invention particularly concerns a new method of producing multimeric fusion proteins involving the TNF superfamily (TNFSF) members as fusion proteins with SPD, and more specifically, CD40L-SPD fusion proteins and useful modifications thereof.
2. Description of Related Art
Numerous proteins can be made using modern molecular biology techniques and used in diagnostic and therapeutic applications. Using recombinant DNA techniques, the DNA encoding a single amino acid chain is constructed and then introduced into a cell which manufactures the final protein. Some cells, especially bacteria like E. Coli, lack the ability to properly fold the amino acid chains into the proper quaternary structure and they often fail to apply the necessary modifications (e.g., glycosylation and disulfide bond formation) that are needed for the protein to be bioactive and resistant to degradation in vivo. While most of these challenges can be met by expressing the amino acid chain in eukaryotic cells like yeast or mammalian cells in vitro, it is not always straightforward to express proteins that consist of two or more amino acid chains. In general, for multichain proteins, the single amino acid chains must associate together in some way either within the producer cell or subsequently after the monomers are secreted from the producer cell. For artificially constructed molecules, the introduction into a single amino acid chain of an amino acid sequence which causes this chain-to-chain association can be an important step in producing multichain proteins.
One of the most widely used methods of causing two amino acid chains to associate is to conjoin, at the DNA coding level, segments from the protein of interest and a segment from a spontaneously dimerizing protein. The best example is to conjoin or fuse a protein with the Fc portion of immunoglobulin, creating a dimeric Fc fusion protein (Fanslow et al., J. Immunol. 136:4099, 1986). A protein of this type can be formed from the extracellular domain of a tumor necrosis factor receptor fused to Fc (termed etanercept and marketed as ENBREL®, a dimeric fusion protein consisting of the extracellular ligand-binding portion of the human 75 kDa (p75) tumor necrosis factor receptor (TNFR) linked to the Fc portion of human IgG1), which is effective in the treatment of rheumatoid arthritis. A second example is the construction of a fusion protein between the dimerizing extracellular portion of CD8 with the extracellular portion of CD40L (Hollenbaugh et al., EMBO J. 11:4313, 1992). Here, the dimerizing CD8 portion of the fusion protein helps to maintain the CD40L portion in the trimeric form needed for its bioactivity. A more recent example is the addition of an isoleucine zipper motif to CD40L, which permits the production of trimeric soluble CD40L molecules (Morris et al., J. Biol. Chem. 274:418, 1999).
The TNF superfamily (TNFSF) consists of an expanding number of proteins (see Table I) which are crucial for the development and functioning of the immune, hematological, and skeletal systems. TNFSF proteins are ligands for a corresponding set of receptors of the TNF receptor superfamily (TNFRSF). All TNFSF members are expressed as Type II membrane proteins, with the exception of lymphotoxin-alpha which is produced as a secreted protein. However, soluble forms of several TNFSF proteins can be released from the cell surface by proteolytic cleavage, usually by specific metalloproteinases.
The production of soluble forms of TNFSF proteins has been an important step in the study of these proteins. Soluble TNFSF ligands can be used to study the activities of these proteins in vitro without the complexities in interpretation that result when cells or cellular membranes expressing TNFSF proteins are added. In addition, soluble forms of several TNFSF proteins have potential as therapeutic agents for human diseases. In particular, TNF-α has been extensively studied for the treatment of cancer and soluble CD40L is currently undergoing clinical trials to assess its antitumor effects.
To produce soluble forms of TNFSF proteins, either the membrane protein is expressed in a cell line possessing a protease capable of separating the TNFSF extracellular domain from the transmembrane domain or a truncated form of the TNFSF protein is produced which consists solely of the extracellular domain plus a signal sequence. In either case, certain soluble forms of TNFSF proteins are unstable in solution as simple homotrimers composed solely of the extracellular domain. For example, naturally solubilized TNF-α is labile under physiological conditions [Schuchmann, 1995 #129]. To solve this stability problem, chimeric proteins have been constructed according to one of four different design principles: (1) The extracellular portion of the TNFSF protein has been expressed fused to the dimeric portion of the immunoglobulin Fc fragment U.S. Pat. No. 5,155,027, Oct. 13, 1992, issued to, Andrzej Z. Sledziewski, et al. In the case of CD40L and OX40L, this yields a soluble molecule which is significantly less active than the native membrane form of this protein. (2) The extracellular portion of the TNFSF protein has been expressed with an antigenic tag (usually the FLAG motif) fused to its N-terminus [Mariani, 1996]. The addition of an antibody to the tag (e.g., anti-FLAG antibody) aggregates these proteins into a multimeric form. Crosslinking enhances activity on B cells. (3) The extracellular portion of the TNFSF protein has been expressed fused to the spontaneously dimerizing extracellular portion of the CD8 molecule [Hollenbaugh, 1992]. In the case of CD40L, this creates a hexameric molecule [Pullen, 1999] which is likely formed by two CD40L trimers attached to three CD8 dimeric stalks. Despite this, the addition of an anti-CD8 antibody to crosslink the CD40L-CD8 fusion protein yields a further enhancement of CD40L activity on B cells. (4) The extracellular portion of the TNFSF protein has been expressed fused to a trimerizing isoleucine zipper which maintains the overall trimeric structure of the protein [U.S. Pat. No. 5 716,805, Feb. 10, 1998, issued to Subashini Srinivasan et al]. This soluble CD40L trimer or ‘sCD40LT’ is the form of that protein now being clinically tested in humans for its anti-tumor effects.
Compounding the difficulties in producing stable forms of soluble TNFSF proteins are compromises in bioactivity. As exemplified by FasL, TNF, and CD40L, many of the soluble forms of these proteins lack the full range of stimulatory activities displayed by the membrane forms of these molecules. For FasL, several groups have reported that naturally produced soluble FasL (generated by proteolytic cleavage from the membrane form) has a spectrum of activities that is distinctly different from the membrane form. Soluble FasL induces apoptosis in activated CD4+ T cells but not fresh, resting CD4+ T cells. In contrast, both types of CD4+ T cells are killed by membrane FasL or a recombinant soluble form of FasL (WX 1) that spontaneously aggregates into oligomers larger than a decamer. For TNF, T cell activation through stimulation of TNFR II, the 80 kDa receptor for TNF, is much greater with membrane TNF′ than soluble TNF. However, if soluble TNF is produced as a tagged protein and crosslinked with an antibody against the tag, then it completely mimics the activities of membrane TNF [Schneider, 1998]. Finally, for CD40L, the stimulatory effects of a soluble form of this TNFSF protein are enhanced by crosslinking [Kehry, 1994] and yields an activity similar to membrane CD40L. For example, soluble CD40L-CD8 fusion protein requires crosslinking with a antibody to CD8 in order to drive resting B cells to proliferate to a degree similar to membrane-bound CD40L.). Even more strikingly, although membrane-bound CD40L expressed on baculovirus-transduced SF9 insect cells is a strong B cell stimulus, small vesicles (10-1,500 nm) prepared from the membranes of these cells are less stimulatory. However, ultracentrifugation of these vesicles creates aggregates which have the full activity of the original membrane CD40L protein. This indicates that B cells are more highly stimulated by a large surface of CD40L than they are by a smaller surface expressing this membrane ligand.
Taken together, the above reports suggest that, for some TNFSF/TNFRSF ligand/receptor pairs at least, it is essential to cluster receptors together for full signaling activity. By this interpretation, the efficacy of the membrane forms of FasL, TNF, and CD40L occurs because these ligands can move in the plane of the membrane toward the contact zone with a receptor-bearing responding cell, thereby clustering ligated receptors to form a receptor-dense region of the membrane. This interpretation is further supported by experiments where crosslinking of a soluble TNFSF protein effectively mimics the activity of the membrane form of the protein [Scheider, 1998].
In all of the above examples, no more than three amino acid chains have been caused to associate together. There is a need to produce multimeric protein molecules where more than three amino acid chains are caused to associate into a single soluble molecular complex. An important example comes from studies of CD40L (also called CD 154 or TNFSF5), which is a member of the TNF family of molecules that are normally expressed as insoluble, cell membrane proteins. It has been shown that soluble homotrimers composed of the extracellular regions of CD40L, TNF, and FasL are not potently active on resting cells that bear receptors for these proteins. However, if these proteins are expressed with a tag on their ends (e.g., the FLAG peptide sequence) and then the trimers are extensively crosslinked using an antibody to FLAG, full activity appears (Schneider et al., J. Exp. Med. 187:1205, 1998). From this, it can be inferred that the soluble single-trimer forms of these molecules does not duplicate the multivalent interactions that normally occur when a receptor-bearing cell comes in contact with the membrane of a cell expressing numerous ligand trimers on its surface. This distinction may be due to a need for receptor clustering for full signaling (Bazzoni and Beutler, N. Engl. J. Med. 334:1717, 1996), which in turn is only possible with a multimeric ligand engaging many receptors at the same time in a localized region of the cell membrane.